analysis of chemical compounds of agarwood oil

IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
ANALYSIS OF CHEMICAL COMPOUNDS OF AGARWOOD
OIL FROM DIFFERENT SPECIES BY GAS
CHROMATOGRAPHY MASS SPECTROMETRY (GCMS)
YUMI Z.H-Y. HASHIM AND N.I. ISMAIL AND P. ABBAS
Department of Biotechnology Engineering, Faculty of Engineering,
International Islamic University Malaysia,
PO Box 10, 50728, Kuala Lumpur, Malaysia.
yumi@iium.edu.my
ABSTRACT: Agarwood oil is a highly prized type of oil due to its unique aroma. The
oil is extracted from the fragrant resin found in the agarwood tree (trunk). The unique
aroma and quality of agarwood resin and oil are contributed by the presence of certain
chemical compounds. In this work, analysis and comparison of the chemical compounds
of agarwood oil from A. malaccensis, A. sub-integra and a mixture of both were
conducted. The essential oils were diluted in hexane (5%) prior to gas chromatography
mass spectrometry (GCMS) analysis performed using Agilent GCMS 7890A coupled
with MSD quadrupole detector 5975 C. Separation of analytes by gas chromatography
was carried out using a Hewlett Packard HP-5MS silica capillary column (30 m X 0.25
mm X 0.25 mm). A total of 107 compounds were identified from the three samples of
agarwood oils. Fifty-five (55) components were identified in A. malaccensis sample
which contributes to the largest portion of the total compounds. About 20% of the
compounds identified were aromatic and sesquiterpenes, which have been revealed to be
the main active compounds of agarwood oils which also give the aroma and pleasant
odour of agarwood. Different compositions or profile of chemical components were
found in agarwood oils from the two different species. Two compounds were commonly
identified in all three samples, namely 3-phenyl-2-butanone and alpha-cubebene. Further
studies are needed to refine the results which later can be used to assist detection and
authentication of agarwood as well as its scientific-based grading.
ABSTRAK: Minyak gaharu merupakan sejenis minyak beraroma unik yang
mendapat permintaan tinggi dan mahal. Minyak ini diekstrak daripada resin
beraroma yang terbentuk di dalam batang pokok gaharu. Keunikan aroma dan
kualiti resin dan minyak gaharu ini bergantung kepada kehadiran bahan kimia
tertentu. Penyelidikan ini menjurus kepada analisis dan perbandingan bahan-bahan
kimia yang terdapat dalam minyak gaharu daripada spesis A. malaccensis, A. subintegra dan campuran kedua-duanya. Minyak gaharu dilarutkan di dalam 5%
heksana sebelum dianalisis menggunakan kromatografi gas Agilent GCMS 7890A
berserta spektormetri jisim (MSD quadrupole detector 5975 C). Kolum kapilari silika
Hewlet Packard HP-5MS (30 m X 0.25 mm X 0.25 mm) digunakan untuk pemisahan
bahan-bahan kimia tersebut. Sejumlah 107 kompaun dikenalpasti dalam ketiga-tiga
sampel minyak gaharu tersebut dengan 55 kompaun didapati di dalam sample A.
malaccensis. Ini merupakan bahagian terbesar daripada keseluruhan kompaun. Sebanyak
20 % daripada keseluruhan kompaun yang dikenalpasti adalah daripada kumpulan
seskuiterpena dan bersifat aromatik, juga merupakan kumpulan yang bertanggungjawab
memberi aroma yang unik kepada minyak gaharu. Setiap spesis didapati mempunyai
komposisi atau profil bahan kimia yang tersendiri. Dua kompaun iaitu 3-phenyl-2butanone dan alpha-cubebene ditemui dalam ketiga-tiga sample yang dianalisis. Kajian
lanjut adalah perlu untuk memurnikan dapatan hasil penyelidikan yang berpotensi bagi
membantu pengenalpastian gaharu berserta pembangunan penggredan gaharu yang lebih
saintifik.
55
IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
KEYWORDS: agarwood oil; A. Malaccensis; A. sub-integra; chemical compounds,
GCMS
1. INTRODUCTION
Agarwood oil is a highly prized type of oil due to its unique aroma. The oil is
extracted from the fragrant resin found in the agarwood tree trunk. The formation of
resin can be stimulated through natural infection of a consortium of microorganisms,
chemical induction and or artificial wounding. Since the formation of resin through
natural infection is a long process with very low yield, this lends to its high price.
The unique aroma and quality of the agarwood resin and oil are contributed by
the presence of certain chemical compounds. Sesquiterpenes have been reported to be
the main active components that play important roles in giving the aroma and
pleasant odor of agarwood [1, 2]. However, there are many other chemical
compounds in the agarwood resin and oil; some of which have medicinal values.
Earlier studies have focused on identification of the chemical compounds found in
specific species of agarwood. For instance, sesquiterpenes were reported to be found
in A. agallocha [3, 4]. There are fifteen species in the Aquilaria genus and eight are
known to produce agarwood [5]. In theory, all members in Aquilaria genus can
produce agarwood; however, until recently it was primarily produced from Aquilaria
malaccensis. Other members of the genus that are usually harvested are A. crassna,
A. sub-integra, A. agallocha and A. sinensis [6, 7].
In this work, analysis and comparison the chemical compounds of agarwood oil from
A. malaccensis and A. sub-integra were conducted. The former is a local species found in
Malaysia; which has connotation with superior quality of resin and oil [6]. Meanwhile, the
latter, with characteristic of faster growth, is more common in Thailand and Cambodia
where the species have been commercially cultivated in plantations. The characteristics of
aroma between species may help in identification and authentication of agarwood as well
as determination of its quality.
2. MATERIALS AND METHODS
2.1 Sample Preparation
Three types of agarwood oils produced using hydrodistillation technique were
obtained from different sources. The samples were labeled sample A (A. malaccensis from
Malaysia), sample B (A. sub-integra from Thailand) and sample C (mixture of A.
malaccensis and A. sub-integra from Malaysia, Grade C) respectively. The essential oils
were diluted in hexane (5%) prior to GCMS analysis.
2.2 Gas Chromatography Mass Spectrometry (GCMS) Analysis
GCMS analysis was performed with a gas chromatography system; Agilent 7890A
(Agilent Technologies) coupled with MSD quadrupole detector 5975 C. Separation of
analytes by gas chromatography was carried out using a Hewlett Packard HP-5MS silica
capillary column (30 m X 0.25 mm X 0.25 mm). The analytical conditions for GCMS are
listed in Table 1.
The peaks from GCMS were detected based on the total ion chromatography (TIC)
and mass chromatograms; and further identified using National Institute of Standards and
Technology (NIST) 2008 mass spectral library.
56
IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
Table 1: Analytical conditions for GCMS analysis (based on [6]).
Program
Conditions
Oven Program
80 °C for 2 min, then 10 °C/min to
250°C for 10 min
Carrier gas
Helium
Gas flow
2 ml/min
Split Ratio
1:50
Injection Volume
1 µl
Mode
Splitless
Interface temperature
250 °C
Electron impact (emission
current)
70 eV
Scan range
32 to 500 amu
3. RESULTS AND DISCUSSION
Examination of three samples of agarwood oils from different sources showed some
variations in GC profile and chemical components. The variations of GC profiles are
shown in Fig. 1 (A-C).
1
2
(a)
57
IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
3
1
2
(b)
3
1
2
(c)
Fig. 1: Total ion chromatogram (TIC) for agarwood oil from different species.
(A) Sample A (A. malaccensis from Malaysia), (B) Sample B (A. sub-integra from
Thailand) and (C) Sample C (mixture for A. malaccensis and A. sub-integra from
Malaysia, Grade C). Labeled peaks are 3-phenyl-2-butanone (1), alpha-cubebene (2),
and globulol (3).
58
IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
A total of 107 compounds were identified from the three samples of agarwood oils.
Fifty five components were identified in sample A which contributes to the largest portion
of the total compounds. In sample B and sample C, there were 35 and 37 compounds
identified respectively. About 20% of the compounds identified were aromatic and
sesquiterpenes which have been revealed to be the main active compounds of agarwood
oils. These main active components play important roles in giving the aroma and pleasant
odor of agarwood [1, 2].
Two compounds were commonly identified in all three samples which are 3-phenyl2-butanone and alpha-cubebene, a waxy herbal odor type with medium odor strength.
Common occurrence of 3-phenyl-2-butanone, was observed in all agarwood oil (A.
malaccensis) obtained from different sources in Peninsular Malaysia [8]. In an earlier
study, benzylacetone was also found to be major compounds found in A. malaccensis [9].
Similar compound named 1,1,4,7-Tetramethyldecahydro-1H-cyclopropa[e]azulen-4ol (also known as globulol) was identified to be present in sample B and sample C. There
were 11 similar compounds identified from sample A and sample C but not sample B.
Most of these compounds were sesquiterpenes such as alpha-caryophyllene (alphahumulene), alpha-calacorene. To this end, this suggests that the compounds could be
potential characteristic compounds of each agarwood species. Nevertheless, further studies
are needed to refine and validate these early findings.
There are several factors that may result in different chemical compositions of
agarwood essential oil which include (a) species/origin of the agarwood tree, (b) methods
of stimulation of agarwood resin, and (c) extraction method of agarwood oil. Our results
showed that different species have different composition of chemical compounds. In
another study, several sesquiterpenes were found in five different types of Chinese
eaglewood essential oil [10].
The quality of agarwood oil is also affected by the stimulation method to produce the
resin. Resin production can be stimulated either through natural infection, chemical
induction or artificial screw techniques which wounded the trunk (nail inserting, holing
and trunk breaking). Several studies have been done to compare the quality of the mantreated and wild agarwood. Chen et al. [1] investigated the chemical compositions of
agarwood oil originating from resin stimulated by chemical methods. Meanwhile, Bhuiyan
et al. [11] examined the difference in composition of oil obtained from naturally infected
tree and artificially inoculated eaglewood (A. agallocha Robx.) by GCMS. The
composition of inoculum, natural variant and specific host-pathogen relationship also play
great role in inducing the resin production in agarwood [11] which relates to the quality
and differential composition of chemical compounds in its resin and oil.
Further, extraction method of agarwood oil also influences the quality and chemical
composition of the oil. Various techniques have been used for agarwood oil extraction:
water distillation (hydrodistillation), steam distillation, solvent extraction and supercritical
fluid extraction [12]. Each technique has its own advantages and disadvantages. In a study
done by Pornpunyapat et al. [13], extraction of agarwood oil was performed by
hydrodistillation at different temperature: 80°C, 100°C and 120°C. In their findings,
hydrodistillation of essential oil at high temperature (120°C) gave the maximum oil yield
and have better physical properties and attributes. Meanwhile, there was significant
difference in the quantity of each composition and types of major components of
agarwood oil extracted using supercritical fluid extraction as compared to distillation
method [2].
59
IIUM Engineering Journal, Vol. 15, No. 1, 2014
Hashim et al.
4. CONCLUSION
In conclusion, using GCMS, we have identified different compositions of chemical
components in agarwood oils from different species namely A. malaccensis and A. subintegra with two compounds commonly identified in all three samples which are 3phenyl-2-butanone and alpha-cubebene. Further studies are needed to refine the results
which later can be used to assist detection and authentication of agarwood as well as its
scientific-based grading.
ACKNOWLEDGEMENT
This research was jointly funded by IIUM Special Grant and Kayu Gaharu (M) Sdn. Bhd.
We would like to thank everyone who contributed to this research.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Chen, H., Yang, Y., Xue, J., Wei, J., Zhang, Z., Chen, H. “Comparison of compositions and
antimicrobial activities of essential oils from chemically stimulated agarwood, wild
agarwood and healthy Aquilaria sinensis (Lour.) gilg trees.” Molecules 16.6 (2011): 488496.
Wetwiyaklung, P., Thavanapong, N., Charoenteeraboon, J. “Chemical constituents and
antimicrobial activity of essential oil and extracts of heartwood of Aquilaria crassna
obtained from water distillation and supercritical fluid carbon dioxide extraction.” Silpakorn
U Science & Tech Journal, 3.1(2009):25-33.
Ishihara, M., Tsuneya, T., Uneyama, K. Guaiane sesquiterpenes from agarwood.
Phytochemistry, 30.10(1991):3343-347.
Nakanishi, T., Yamagata, E., Yoneda, K., Nagashima, T., Kawasaki, I., Yoshida, T., Mori,
H., Miura, I. Three fragrant sesquiterpenes of agarwood. Phytochemistry, 23.9(1984):206667.
Eurlings, M.C.M., Gravendeel, B. TrnL-trnF sequence data imply paraphyly of Aquilaria
and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification.
Pl. Syst. Evol., 254(2005):1-12.
Ng, L.T., Chang, Y.S., Azizol, A.K. A review on agar (gaharu) producing Aquilaria species.
J. Trop. Forest Products, 2.2(1997):272-85.
Burkill, I.H. A dictionary of the economic products of the Malay Peninsula. Ministry of
Agriculture, Kuala Lumpur. 1(1966):198-206.
Nor Azah MA., Chang, YS., Mailina J., Abu Said A., Abd Majid J., Saidatul Husni S., Nor
Hasnida H., Nik Yasmin YComparison of chemical profile of selected gaharu oils from
Peninsular Malaysia. The Malaysian Journal of Analytical Sciences. 12.2(2008):338-40.
Yaacob, K.B., Joulain, D. Volatile constituents from infected wood of Aquilaria malaccensis
Benth. Sains Malaysiana, 29(2000):197-200.
Mei, Wen-Li., Zeng, Yan-Bo., Wu, J., Cui, Hai-Bin., Dai, Hao-Fu. Chemical composition
and anti-MRSA activity of the essential oil from Chinese eaglewood. Journal of Chinese
Pharmaceutical Sciences, 17(2008):225-29.
Bhuiyan, M.N.I., Begum, J., Bhuiyan, M.N.H. Analysis of essential oil of eaglewood tree
(Aquilaria agallocha Roxb.) by gas chromatography mass spectrometry. Bangladesh J
Pharmacol, 4(2009):24-28.
Naef, R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of
Aquilaria species: A review. Flavour Frag J., 26(2011):73-87.
Pornpunpayat, J., Chetpattananondh, P., Tongurai, C. Mathematical modeling for extraction
of essential oil from Aquilaria crassna by hydrodistillation and quality of agarwood oil.
Bangladesh J Pharmacol, 6(201):18-24.
60